Nucleic acid based fluorescent sensor for mercury detection

ABSTRACT

A nucleic acid enzyme comprises an oligonucleotide containing thymine bases. The nucleic acid enzyme is dependent on both Hg 2+  and a second ion as cofactors, to produce a product from a substrate. The substrate comprises a ribonucleotide, a deoxyribonucleotide, or both.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to provisional application No. 60/955,316 entitled “Nucleic Acid Based Fluorescent Sensor For Mercury Detection” filed 10 Aug. 2008, attorney docket no. ILL05-101-PRO, the entire contents of which are hereby incorporated by reference, except where inconsistent with the present application.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This subject matter of this application may have been funded in part under the following research grants and contracts: National Science Foundation Contract Numbers CTS-0120978 and DMI-0328162, and U.S. Department of Energy Contract Number DE-FG02-01-ER63179. The U.S. Government may have rights in this invention.

BACKGROUND

Mercury is a highly toxic heavy metal in the environment. Mercury exposure can cause a number of severe adverse health effects, such as damages in the brain, nerve system, immune system, kidney, and many other organs.^([1]) Mercury contamination comes from both nature and human activities, and an annual releasing of 4,400 to 7,500 metric tons of mercury to the environment was estimated by the United Nations Environment Programme (UNEP).^([2]) Therefore, highly sensitive and selective mercury sensors are very useful in understanding its distribution and pollution and in preventing mercury poisoning. Towards this goal, many fluorescent small organic molecule-based Hg²⁺ sensors have been reported, which change their emission properties upon binding to Hg²⁺. Most of these sensors, however, require the involvement of organic solvent, show quenched emissions, and suffer from poor selectivity.^([3-11]) Only a few such sensors can detection Hg²⁺ in water with high sensitivity and selectivity.^([12-17]) Hg²⁺ sensors based on foldamers^(,[18, 19]) oligonucleotides,^([20]) genetically engineered cells,^([21]) enzymes,^([22]) antibodies,^([23]) transcriptional regulatory proteins,^([24,25]) DNAzymes,^([26]) and chemically modified optical fibers^([27,28]) capillary optode,^([29,30]) membranes,^([31]) electrodes,^([32,33]) mesoporous silica,^([34]) and nanoparticles^([35]) are also known. For environmental monitoring applications, such as detection of Hg²⁺ in drinking water, a detection limit of lower than 10 nM (the toxic level defined by the US Environmental Protection Agency (EPA)) is required. However, few reported mercury sensors can reach such sensitivity.^([11,21,25]) We are interested in using catalytic DNA or DNAzymes to design metal sensors that can achieve the goal.^([36,37])

DNAzymes are DNA-based biocatalysts.^([38-42]) Similar to protein enzymes or ribozymes, DNAzymes can also catalyze many chemical and biological transformations, and some of the reactions require specific metal ions as cofactors. Highly effective fluorescent and colorimetric sensors have been demonstrated for Pb²⁺ and UO₂ ²⁺ with DNAzymes.^([36,37,43]) These sensors showed picomolar to low nanomolar sensitivity and thousand to million-fold selectivity. In the presence of target metal ions, the fluorescence enhancement was generally greater than 10-fold, and signal generation took only 2 min or less. These sensors can be used at room temperature in aqueous solutions and no organic solvents are needed. Recently, DNAzyme-based electrochemical metal sensors are also reported.^([44]) Compared to protein or RNA, DNA is relatively more cost-effective to produce and more stable. DNAzymes can be denatured and renatured many times without losing their activities.^([39]) Therefore, DNAzymes are useful in metal detection.

It was reported that Hg²⁺ can specifically bind in between two DNA thymine bases and promote such T-T mismatches into stable base pairs (FIG. 1 d).^([20,45,46]) This property was applied by Ono and co-workers to design a fluorescent sensor for Hg²⁺ detection.^([20]) The sensor consisted of a single-stranded thymine rich DNA with the two ends labeled with a fluorophore and a quencher, respectively. In the presence of Hg²⁺, the two ends were brought close to each other, resulting in decreased fluorescence. A detection limit of 40 nM was reported.^([20]) Being sensitive and selective, this sensor is a “turn-off” sensor and fluorescence intensity decreased in the presence of Hg²⁺, which may give false positive results caused by external quenchers or other environmental factors that can also induce fluorescence decrease. The Hg²⁺ stabilization effects on T-T mismatches have also been applied to design colorimetric sensors with DNA-functionalized gold nanoparticles and a detection limit of 100 nM was achieved.^([35]) In previous DNAzyme work, a signaling method called catalytic beacon was designed in which the metal binding site in DNAzymes and the fluorescence signaling part are spatially separated.^([36,37,47])

SUMMARY

In a first aspect, the present invention is a nucleic acid enzyme, comprising an oligonucleotide containing thymine bases. The nucleic acid enzyme is dependent on both Hg²⁺ and a second ion as cofactors, to produce a product from a substrate comprising a ribonucleotide, a deoxyribonucleotide, or both.

In a second aspect, the present invention is a method of detecting Hg²⁺ in a sample comprising forming a mixture comprising (1) a nucleic acid enzyme, (2) the sample, (3) a substrate, and (4) a second ion, to produce a product from the mixture; and determining the presence of the product. The enzyme comprises at least one quencher and is dependent on both the Hg²⁺ and the second ion as cofactors to produce the product from the substrate. Furthermore, the substrate comprises a ribonucleotide, a deoxyribonucleotide, or both, and the substrate comprises at least one fluorophore and at least one quencher.

In a third aspect, the present invention is a method of determining the concentration of Hg²⁺ in the presence of other ions, in a sample, comprising forming a mixture comprising (1) a nucleic acid enzyme, (2) the sample, (3) a substrate, and (4) a second ion, to produce a product from the mixture; determining the presence of the product; and determining the concentration of the Hg²⁺ by measuring an amount of the product produced. The enzyme comprises at least one quencher and is dependent on the Hg²⁺ and the second ion as cofactors to produce the product from the substrate. Furthermore, the substrate comprises a ribonucleotide, a deoxyribonucleotide, or both, and the substrate comprises at least one fluorophore and at least one quencher.

In a fourth aspect, the present invention is a sensor for Hg²⁺, comprising (1) a nucleic acid enzyme, (2) a substrate, and (3) a second ion. The enzyme comprises at least one quencher and is dependent on both the Hg²⁺ and the second ion as cofactors to produce a product from the substrate. Furthermore, the substrate comprises a ribonucleotide, a deoxyribonucleotide, or both, and the substrate comprises at least one fluorophore and at least one quencher.

A “nucleic acid enzyme” is a nucleic acid molecule that catalyzes a chemical reaction. The nucleic acid enzyme may be covalently linked with one or more other molecules yet remain a nucleic acid enzyme. Examples of other molecules include dyes, quenchers, proteins, and solid supports. The nucleic acid enzyme may be entirely made up of ribonucleotides, deoxyribonucleotides, or a combination of ribo- and deoxyribonucleotides.

A “sample” may be any solution that may contain an ion (before or after pre-treatment). The sample may contain an unknown concentration of an ion. For example, the sample may be paint that is tested. The sample may be diluted yet still remain a sample. The sample may be obtained from the natural environment, such as a lake, pond, or ocean, an industrial environment, such as a pool or waste stream, a research lab, a common household, or a biological environment, such as blood. Of course, sample is not limited to the taking of an aliquot of solution but also includes the solution itself. For example, a biosensor may be placed into a body of water to measure for contaminants. In such instance, the sample may comprise the body of water or a particular area of the body of water. Alternatively, a solution may be flowed over the biosensor without an aliquot being taken. Furthermore, the sample may contain a solid or be produced by dissolving a solid to produce a solution. For example, the solution may contain soil from weapon sites or chemical plants.

“Measuring an amount of the product produced” includes measuring the result of the production of a product by an enzyme. For example, in an embodiment where the substrate comprises a quencher and fluorophore and the enzyme comprises a second quencher, and cleavage of the substrate by the enzyme leads to dissociation of the product from the enzyme, “measuring an amount of the product produced” includes detecting the increase of fluorescence. Thus, the product is measured by detecting its inability to quench fluorescence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (a) The secondary structure of the originally reported UO₂ ²⁺-specific DNAzyme. (b) The new UO₂ ²⁺-specific DNAzyme with the replaced stem loop. One of the A·G mismatches was also replaced by an A-T base pair. (c) The stem loop part of DNAzymes with zero to six T-T mismatches (top). Gel image showing the fraction of cleavage after 1 min reaction time in the absence or presence of 10 μM Hg²⁺ (middle). The ratio of cleavage fraction after 1 min in the presence or absence of Hg²⁺ (bottom). (d) Schematics of a T-T mismatch stabilized by a Hg²⁺ ion.

FIG. 2. (a) The secondary structure and modification of the Hg²⁺ sensor DNAzyme. (b) Schematic presentation of the sensor design. (c) Fluorescence spectra of the sensor (with the DNAzyme and 1 μM UO₂ ²⁺) in the absence and 8 min after addition of 0.5 μM Hg²⁺.

FIG. 3. Sensitivity of the Hg²⁺ sensor. (a) Kinetics of fluorescence increase in the presence of varying concentrations of Hg²⁺. (b) Hg²⁺ dependent fluorescence increase rate. Rates were calculated in the time window of 1-2 min in (a). Inset: sensor responses at low Hg²⁺ concentrations. The y-axis is the fluorescence counts increase per second. The DNAzyme and UO₂ ²⁺ concentrations were 100 nM and 1 μM, respectively.

FIG. 4. Selectivity of the Hg²⁺ sensor. All competing metal ions were tested at 1, 20, and 1000 μM. For comparison, sensor responses to 20, 100, and 500 nM of Hg²⁺ were also presented. The DNAzyme and UO₂ ²⁺ concentrations were 100 nM and 1 μM, respectively.

FIG. 5. Rational design of an allosteric Hg²⁺ DNAzyme based on the 8-17 DNAzyme. (A) the secondary structure of the 8-17 DNAzyme. (B) Schematics of cleavage of the 8-17 DNAzyme by Pb²⁺. (C) Insertion of a stem in the enzyme strand with T-T mismatches can make the DNAzyme into a Hg²⁺ dependent enzyme.

FIG. 6. Rational design of an allosteric Hg²⁺ DNAzyme based on the 10-23 DNAzyme. (A) the secondary structure of the 10-23 DNAzyme. (B) Schematics of cleavage of the 10-23 DNAzyme by Mg²⁺. (C) Insertion of a stem in the enzyme strand with T-T mismatches can make the DNAzyme into a Hg²⁺ dependent enzyme.

DETAILED DESCRIPTION

The present invention makes use of the discovery of new nucleic acid enzymes, which may be used in a sensor system for Hg²⁺, and in a method of determining the concentration of mercury in a sample, especially in the presence of other ions. These new nucleic acid enzymes are designed from existing nucleic acid enzymes. The existing nucleic acid enzymes typically have a secondary structure necessary for activity, maintained by hybridization between bases within the nucleic acid enzyme. By introducing one or more thymine-thymine mismatches, opposite each other and within those hybridized portions of the nucleic acid enzyme which maintain the secondary structure, the secondary structure is destabilized. When Hg²⁺ is present, however, it causes the T-T mismatches to form stable base pairs, stabilizing the secondary structure and restoring activity. These new nucleic acid enzymes require two different ions as cofactors: the original ion and Hg²⁺.

It is also possible to artificially create such secondary structure stabilizing bases in the nucleic acid enzyme by introducing a stem loop having at least two consecutive pairs of hybridizing nucleotides in the stem loop and at least three nucleotides in between; one or two additional nucleotides may also be included at each end of the introduced section. The nucleic acid enzyme can then be made response to Hg²⁺ by introducing one or more pairs of thymine bases, opposite each other and within those hybridized portions of the nucleic acid enzyme which maintain the secondary structure. Again, the result is a nucleic acid enzyme having two different ions as cofactors: the original ion and Hg²⁺.

The present invention provides a simple, rapid, inexpensive, selective and sensitive method for detecting the presence of Hg²⁺, with background fluorescence signal near zero, and is an important and useful tool in preventing or at least lowering health and environmental risks associated with the environmental contaminant Hg²⁺. A nucleic acid enzyme that catalyzes the cleavage of a nucleic acid in the presence of two ions, Hg²⁺ and a second ion, is used. The nucleic acid enzyme may be RNA (ribozyme), DNA (deoxyribozyme), a DNA/RNA hybrid enzyme, or a peptide nucleic acid (PNA) enzyme. PNAs comprise a polyamide backbone and the bases found in naturally occurring nucleosides and are commercially available, e.g., from Biosearch, Inc. (Bedford, Mass.). Nucleic acids including nucleotides containing modified bases, phosphate, or sugars may be used in the compositions and methods of the present invention. Modified bases are well known in the art and include inosine, nebularine, 2-aminopurine riboside, N⁷-denzaadenosine, and O⁶-methylguanosine.^([55]) Modified sugars and phosphates are also well known and include 2′-deoxynucleoside, abasic, propyl, phosphorothioate, and 2′-O-allyl nucleoside.^([55]) DNA/RNA hybrids and PNAs may be used in the compositions and methods of the present invention.

A highly sensitive and selective catalytic beacon for mercury was rationally designed based on a uranium-specific DNAzyme, shown in FIG. 2( a). Hg²⁺ enhanced the DNAzyme activity through allosteric interactions, and a series of allosteric DNAzymes with varying number of thymine-thymine mismatches were tested. The optimal DNAzymes was labeled with fluorophore and quenchers to construct a catalytic beacon. The sensor has a detection limit of 2.4 nM, which is lower than the EPA limit of Hg²⁺ in drinking water. It is also highly selective and is silent to any other metal ions with up to millimolar concentration levels. The catalytic beacon performance may be further improved by incorporation of in vitro selections to optimize the allosteric interactions.^([52]) This work further demonstrated that DNAzymes are a great platform for metal sensing. Additional nucleic acid enzymes, using the same strategy, have also been designed, and their sequences are shown in the table along with nucleic acid enzyme used to design the highly sensitive and selective catalytic beacon for mercury.

The nucleic acid enzymes and their substrates may be used in a “turn-on” sensor for Hg²⁺. These sensors are similar to those described in U.S. Pat. No. 6,890,719, except that the nucleic acid enzyme requires Hg²⁺ and a second ion as cofactors. For the sensor to be responsive to Hg²⁺, the second ion is included with the sensor. The second ion may be monovalent, divalent, trivalent, or polyvalent. Examples of monovalent cations include K⁺, Na⁺, Li⁺, Tl⁺, NH₄ ⁺ and Ag⁺. Examples of divalent cations include Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Cu²⁺, Pb²⁺, Pt²⁺, Ra²⁺, Ba²⁺, UO₂ ²⁺ and Sr²⁺. Examples of trivalent cations include Co³⁺, Cr³⁺, and lanthanide ions (Ln³⁺). Polyvalent cations include Ce⁴⁺, and Cr⁶⁺. The second ion also includes ions having a metal in a variety of oxidation states. Examples include K(I), Na(I), Li(I), Tl(I), Ag(I), Hg(I), Mg(II), Ca(II), Mn(II), Co(II), Ni(II), Zn(II), Cd(II), Pb(II), Pt(II), Ra(II), Ba(II), Sr(II), Co(III), Cr(III), Ln(III), Ce(IV), Cr(VI) and U(VI).

The sequences of nucleic acid enzymes are indicated in the table below, along with specific examples, and substrates for the enzymes. R, Y, r and n represent purine, pyrimidine, a ribonucleotide and any nucleotide, respectively. F indicates a fluorophore, and Q indicates a quencher. The cofactor(s) for the enzyme is (are) included in the first column. Preferably, the nucleic acid enzyme contains 38 to 100 bases.

Nucleic acid enzyme 5′ CCA TCT CTG CA G TCG G GT AGT SEQ ID 39E (UO₂ ²⁺)(FIG. TAA A CC GAC CTT CAG AC A TAG NO: 1 1(a)) TGA GT 3′ 39S (substrate for 39E; 3′ GGT AGA GAA GGr ATA TCA CTC A 5′ SEQ ID FIG. 1(a)) NO: 2 Nucleic acid enzyme 5′ CCA TCT CTT CA G TTC G GA AA C SEQ ID E_(Hg)0T (UO₂ ²⁺; FIG. GAA C CT TCA GAC ATA GTG AGT 3′ NO: 3 1(b)) Nucleic acid enzyme 5′ CCA TCT CTT CA G TTC G GA AA C SEQ ID E_(Hg)1T (UO₂ ²⁺ and Hg²⁺; GTA C CT TCA GAC ATA GTG AGT 3′ NO: 4 FIG. 1(c)) Nucleic acid enzyme 5′ CCA TCT CTT CA G TTTC G GA AA SEQ ID E_(Hg)2T (UO₂ ²⁺ and Hg²⁺; C GTT A C CT TCA GAC ATA GTG AGT NO: 5 FIG. 1(c)) 3′ Nucleic acid enzyme 5′ CCA TCT CTT CA G TTT TC G GA AA SEQ ID E_(Hg)3T (UO₂ ²⁺ and Hg²⁺; C GTT TA C CT TCA GAC ATA GTG NO: 6 FIG. 1(c)) AGT 3′ Nucleic acid enzyme 5′ CCA TCT CTT CA G TTT TTC G GA SEQ ID E_(Hg)4T (UO₂ ²⁺ and Hg²⁺; AA C GTT TTA C CT TCA GAC ATA NO: 7 FIG. 1(c)) GTG AGT 3′ Nucleic acid enzyme 5′ CCA TCT CTT CA G TTT TTT C G GA SEQ ID E_(Hg)5T (UO₂ ²⁺ and Hg²⁺; AA C GTT TTT A C CT TCA GAC ATA NO: 8 FIG. 1(c)) GTG AGT 3′ Nucleic acid enzyme 5′ CCA TCT CTT CA G TTT TTT TC G SEQ ID E_(Hg)6T (UO₂ ²⁺ and Hg²⁺; GA AA C GTT TTT TA C CT TCA GAC NO: 9 FIG. 1(c)) ATA GTG AGT 3′ Nucleic acid enzyme 5′ CAT CTC TTC TCC GAG CCG GTC SEQ ID 17E (Pb²⁺; FIG. 5A) GAA ATA GTG AGT 3′ NO: 10 17S (substrate for 17E; 3′ GTA GAG AAG GrA TAT CAC TCA 5′ SEQ ID FIG. 5A) NO: 11 Nucleic acid enzyme 5′ CAT CTC TTC TCC GAG CCG GTC SEQ ID 17E_(Hg) (Pb²⁺ and Hg²⁺; GAA ATA TTT TTA GCT GAA AAG CTT NO: 12 FIG. 5C) TTA AAA GTG AGT 3′ 17S_(Hg) (substrate for 3′ GTA GAG AAG GrA TAT nnn CAC SEQ ID 17E_(Hg); FIG. 5C) TCA 5′ NO: 13 Nucleic acid enzyme 6E 5′ GGG CAA GGC TAG CTA CAA CGA SEQ ID (Mg²⁺; FIG. 6A) CTA CGG CAG TC 3′ NO: 14 6S (substrate for 6E; 3′ CCC GUU G GAU GCC GUC AG 5′ SEQ ID FIG. 6A) NO: 15 Nucleic acid enzyme 5′ GGG CAA GGC TAG CTA CAA CGA SEQ ID 6E_(Hg) (Mg²⁺ and Hg²⁺; CTA AAT TTT TAG CTG AAA AGC TTT NO: 16 FIG. 6C) TAA CGG CAG TC 3′ 6S_(Hg) (substrate for 6E_(Hg); 3′ CCC GUU G GAU nnn GCC GUC AG 5′ SEQ ID FIG. 6C) NO: 17 Nucleic acid enzyme 5′ GAA TCG AAC T GTC AGT GAC TCG SEQ ID E2112 (Mn²⁺)^([53]) AAA GCA CGG A 3′ NO: 18 S2112 (substrate for 3′ CTT AGC TTGG n nr TCG TGCCT 5′ SEQ ID E2112)^([53]) NO: 19 Nucleic acid enzyme 5′ GAA TCG AAC T GTTTTC AGT SEQ ID E2112_(Hg(a)) (Mn²⁺ and GTTATC TCG AAA GCA CGG A 3′ NO: 20 Hg²⁺) Nucleic acid enzyme 5′ GAA TCG AAC T GTTTTTC AGT SEQ ID E2112_(Hg(b)) (Mn²⁺ and GTTATTC TCG AAA GCA CGG A 3′ NO: 21 Hg²⁺) Nucleic acid enzyme 5′ nnn YYY YYY Y AGT T_(m) YR nnnn YR SEQ ID E_(Hg)XT (UO₂ ²⁺ and Hg²⁺) T_(m) AC CT TCA GAC A YRR YRR nn 3′ NO: 22 (consensus sequence) (m = 1-6) Nucleic acid enzyme 5′ nnY YYY YYY TCC Gnn nCG GTC SEQ ID 17E_(Hg(x)) (Pb²⁺ and Hg²⁺) GAA A TA TT T_(p) A GC T nnnn AG CT T_(p) NO: 23 (consensus sequence) A A nn RYR RRY 3′ (p = 1-6) Nucleic acid enzyme 5′ RRR YR A GGC TAG CTA CAA CGA SEQ ID 6E_(Hg(x)) (Mg²⁺ and Hg²⁺) C TA AAT T T_(q) AG CT nnnn AGC TT_(q) AA NO: 24 (consensus sequence) YRR YRR nn 3′ (q = 1-6)

F indicates a fluorophore, and Q indicates a quencher. Essentially any fluorophore may be used, including BODIPY, fluoroscein, fluoroscein substitutes (Alexa Fluor dye, Oregon green dye), long wavelength dyes, and UV-excited fluorophores. These and additional fluorophores are listed in Fluorescent and Luminescent Probes for Biological Activity. A Practical Guide to Technology for Quantitative Real-Time Analysis, Second Ed. W. T. Mason, ed. Academic Press (1999)^([56]). In preferred embodiments, the fluorophore is 6-carboxyfluorescein (FAM). FAM has an excitation range of 460-500 nm.

Other fluorophores included quantum dots and silica nanoparticles. Each type of quantum dot displays a unique emission wavelength. Preferred quantum dot particles include quantum dot semiconductors, such as CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, PIn, and PbSe. Additional preferred quantum dots may include ternary quantum dots, such as Cd_(x)Zn_(1-x)Se or CdS_(x)Se_(1-x). Additional preferred quantum dots may include core-shell quantum dots, such as those having a CdSe core and ZnS shell. The quantum dots can also have different morphologies, including dots, rods, tetrapods, and the like. In a preferred aspect, the particles are quantum dot semiconductors having average diameter from 2 to 50 nanometers.

A quencher is a molecule that absorbs the energy of the excited fluorophore. Close proximity of a fluorophore and a quencher allow for the energy to be transferred from the fluorophore to the quencher. By absorbing this energy, the quencher prevents the fluorophore from releasing the energy in the form of a photon, thereby preventing fluorescence.

Quenchers may be categorized as non-fluorescent and fluorescent quenchers. Non-fluorescent quenchers are capable of quenching the fluorescence of a wide variety of fluorophores. Generally, non-fluorescent quenchers absorb energy from the fluorophore and release the energy as heat. Examples of non-fluorescent quenchers include 4-(4′-dimethylaminophenylazo)benzoic acid) (Dabcyl), QSY-7, and QSY-33.

Fluorescent quenchers tend to be specific to fluorophores that emit at a specific wavelength range. Fluorescent quenchers often involve fluorescence resonance energy transfer (FRET). In many instances the fluorescent quencher molecule is also a fluorophore. In such cases, close proximity of the fluorophore and fluorescent quencher is indicated by a decrease in fluorescence of the “fluorophore” and an increase in fluorescence of the fluorescent quencher. Commonly used fluorescent fluorophore pairs (fluorophore/fluorescent quencher) include fluorescein/tetramethylrhodamine, IAEDANS/fluorescein, fluorescein/fluorescein, and BODIPY FL/BODIPY FL.

When choosing a fluorophore, a quencher, or where to position these molecules, it is important to consider, and preferably to test, the effect of the fluorophore or quencher on the enzymatic activity of the nucleic acid enzyme. Also, it is preferable that the fluorophore display a high quantum yield and energy transfer efficiency. Long-wavelength (excitation and emission) fluorophores are preferred because of less interference from other absorbing species. The fluorophore should also be less sensitive to pH change or to non-specific quenching by metal ions or other species.

Methods and devices for detecting fluorescence are well developed. Essentially any instrument or method for detecting fluorescent emissions may be used. For example, WO 99/27351 describes a monolithic bioelectrical device comprising a bioreporter and an optical application specific integrated circuit (OASIC).^([57]) The device allows remote sampling for the presence of substances in solution. Furthermore, the fluorescence may be measured by a number of different modes. Examples include fluorescence intensity, lifetime, and anisotropy in either steady state or kinetic rate change modes.^([58])

Sometimes other factors in a solution such as pH, salt concentration or ionic strength, or viscosity will have an effect on fluorescence, and may even affect the hybridization of the substrate and enzyme. Therefore, in preferred methods, controls are run to determine if the solution itself, regardless of enzymatic activity, is altering the fluorescence. Such controls include the use of non-cleavable substrates and or substrate without the presence of enzyme.

The sensor system may be used to determine an amount of mercury in a sample, such as a water sample, a biological sample (such as blood or serum), or a solid sample, such as soil or paint. Preferably, a solid sample is first dissolved into solution. The samples may be diluted or concentrated prior to testing, and may also be buffered. The sensor system is able to determine an amount of mercury in the presence of other ions, preferably other metal ions, such as Mg²⁺, Ca²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn^(2÷), Cd²⁺, Pb²⁺, UO₂ ²⁺ and Tb³⁺, or in the presence of other ions.

In light of the present disclosure, one of ordinary skill in the art would know how to modify the nucleic acid biosensors to include nucleic acid enzymes. For example, a biosensor of the present invention may comprise a nucleic acid enzyme labeled with a fluorescent quencher, a substrate labeled with a fluorophore and a second fluorescent quencher, and a device to detect fluorescence such as a fluorescence microscope or a fluorometer. In a method using this embodiment, the enzyme and substrate are contacted with a sample suspected of containing an ion to which the enzyme is sensitive. Fluorescence is measured and compared to a control wherein the ion is absent. Change in fluorescence is indicative of the presence of the ion.

Of course, many variants of even this simple embodiment are included within the scope of the invention. Such variants include placing the enzyme, substrate, and sample in the well of a microtiter plate and measuring fluorescence with a microtiter plate reader. In another variation, the enzyme is attached to a solid support. When the enzyme is attached to a solid support, it is preferable that a linker is used. An exemplary linking system is biotin/streptavidin. For example, the biotin molecule may be linked to the enzyme and a plate may be coated with streptavidin. When linking an enzyme to a solid support, it is important to determine the effect of linkage on the enzymatic activity of the enzyme.

In an alternative embodiment, the solid support may be a bead and fluorescence measured using a flow cytometer. In embodiments having the enzyme attached to a solid support, the biosensor may be reusable. Old substrate and sample is removed, leaving the enzyme in place. New substrate and sample may then be added.

Recently, a UO₂ ²⁺-specific DNAzyme was isolated by in vitro selection.^([37]) The secondary structure of the DNAzyme is shown in FIG. 1 a, which contained a substrate strand (39S) and an enzyme strand (39E). 39S has a single RNA linkage (rA) that serves as the cleavage site. 39E binds 39S through two substrate binding arms. The catalytic core in 39E contains a stem loop and an eight-nucleotide bulge. Further studies indicated that the exact nucleotide sequence in the stem loop was unimportant for activity, as long as such a structure was maintained. For example, by replacing the stem loop to that shown in FIG. 1 b, the DNAzyme was still active. In addition to the change made to the stem loop, one of the A·G mismatches in the left substrate binding arm away from the cleavage site in FIG. 1 a was replaced by a A-T Watson-Crick base pair, while the other A·G mismatch close to the cleavage site was maintained. The new enzyme strand was then named E_(Hg)0T (FIG. 1 b), which was used as a scaffold to engineer allosteric DNAzymes that can detect Hg²⁺. In addition to having such a replaceable stem loop, the uranium DNAzyme was chosen for Hg²⁺ sensing also for the following reasons. First, this DNAzyme is active only in the presence of UO₂ ²⁺ and 1 μM UO₂ ²⁺ is sufficient to saturate its activity.^([37]) Unlike other common metal ions, UO₂ ²⁺ does not present in high concentrations in most environmental samples. Therefore, if the sensor system is saturated with UO₂ ²⁺, external metals are unlikely to interfere with the detection. Even though uranium is a radionuclide, 1 μM UO₂ ²⁺ does not cause health or environmental concerns because uranium is ubiquitous in the environment, and even in drinking water, 130 nM uranium is allowed, according to the US EPA. Since the sensing application requires only 500 μL or less sensor samples, the environmental impact is negligible. Second, the enzyme kinetics is fast. The E_(Hg)0T DNAzyme shown in FIG. 1 b has a rate constant of 2.0 min⁻¹ in the presence of 1 μM UO₂ ²⁺, which allows fast sensor response. Finally, the DNAzyme is relatively small in size and can be chemically synthesize and modified with high yield.

To incorporate Hg²⁺ recognition elements into the DNAzyme, using rational design methods by introducing one to six T-T mismatches in stem region of E_(Hg)0T (FIG. 1 c). All other nucleotides were kept the same. The sequence of E_(Hg)0T was designed in such a way that no stable secondary structures in the catalytic cores of all the DNAzymes (from E_(Hg)1T to E_(Hg)6T) involving the thymine insertions were predicted by the Mfold program.^([48]) As a result, the DNAzymes cannot fold into their active structures in the absence of Hg²⁺. Addition of Hg²⁺ should quickly fold the DNAzymes into their active conformations without kinetic traps. Because the effect of Hg²⁺ is spatially away from the UO₂ ²⁺ binding site, such DNAzymes belongs to allosteric DNAzymes.^([49])

EXPERIMENTAL DETAILS

To test whether Hg²⁺ can enhance the activity of these thymine rich

DNAzymes, 1 μM of the DNAzyme complexes were incubated with 10 μM Hg²⁺ for 10 min. at room temperature. The substrate strand was labeled with a FAM fluorophore on the 5′-end. UO₂ ²⁺ was added to initiate the cleavage reaction. After 1 min, the reaction was stopped and the samples were loaded onto a 20% denaturing polyacrylamide gel to separate the cleaved and uncleaved substrate. As shown in FIG. 1 c, in all the DNAzymes with T-T mismatches, the fraction of cleavage was higher in the presence of Hg²⁺, suggesting that Hg²⁺ indeed helped to stabilize the stem loop structure and made the DNAzymes more active. For the E_(Hg)1T, E_(Hg)2T, and E_(Hg)3T DNAzymes, the cleavage bands in the absence of Hg²⁺ were also quite clear, suggesting that the DNAzymes may transiently fold into their active conformations even with several T-T mismatches. Such tolerability, however, dropped very quickly as the number of mismatches increases. For each DNAzyme, the ratio of cleavage fraction in the presence and absence of Hg²⁺ was determined (bottom of FIG. 1 c), which approximately represents the fold of activity enhancement caused by Hg²⁺, and this value positively correlates with signal-to-background ratio for sensing applications.

In the above experiment, the DNAzymes were first allowed to equilibrate with Hg²⁺, and UO₂ ²⁺ was added to initiate the reaction. To detect Hg²⁺, it is more desirable to add Hg²⁺ to the DNAzyme/UO₂ ²⁺ mixture to initiate the cleavage reaction. Because E_(Hg)5T and E_(Hg)6T showed the highest activity enhancement by Hg²⁺, the rates of cleavage initiated by adding 10 μM Hg²⁺ to the mixture of 1 μM DNAzyme and 1 μM UO₂ ²⁺ was calculated. Compared to the original DNAzyme E_(Ng)0T, which had a rate constant of 2.0 min⁻¹, the values for E_(Hg)5T and E_(Hg)6T were 0.61 and 0.45 min⁻¹, respectively. Therefore, DNAzymes with more T-T mismatches had lower rates, which could be explained by that it took more time for longer DNA to find the right conformation. As a compromise between the rate of the reaction and the fold of activity enhancement, E_(Hg)5T was chosen for further studies.

The Hg²⁺ catalytic beacon is shown in FIG. 2 a. The original E_(Hg)5T enzyme strand was extended on the 5′-end by five nucleotides, and the substrate was also extended accordingly to form base pairs with the extended enzyme. Such extensions were made to increase the hybridization efficiency between the two strands. To generate signal, a fluorophore (FAM) was labeled on the 5′-end of the substrate, a quencher was labeled on the 3′-end of the enzyme, and an additional quencher was attached on the 3′-end of the substrate. Both quenchers were black hole quenchers. Such dual quencher labeling method gave very low background fluorescence and therefore allowed high signal enhancement.^([50]) The DNAzyme was mixed with UO₂ ²⁺ to become a mercury sensor (FIG. 2 b). In the absence of Hg²⁺, the DNAzyme was incapable of binding UO₂ ²⁺ because of the active secondary structure cannot form. Addition of Hg²⁺ quickly restored the stem loop structure and activated the DNAzyme to cleave the substrate, releasing the fluorophore-labeled piece and giving increased fluorescence. The fluorescence spectra of the sensor before and 8 min after addition of 500 nM Hg²⁺ is shown in FIG. 2 c, and ˜50-fold increase in the 520 nm peak was observed. Such level of fluorescence increase is among the highest in functional nucleic acid based sensors.^([51])

Given the very high fluorescence enhancement, the DNAzyme was titrated with varying concentrations of Hg²⁺ and the kinetics of fluorescence enhancement at 520 nm was monitored. As shown in FIG. 3 a, higher concentrations of Hg²⁺ produced higher rates of emission enhancement. All the kinetic traces showed a roughly linear increase in 1-2 min time window after addition of Hg²⁺ and therefore the rate of fluorescence increase in this window was calculated to quantify Hg²⁺ concentration (FIG. 3 b). The Hg²⁺-dependent response had a sigmoid shape and was fit to a Hill plot with a Hill coefficient of 2.1. This result suggests that Hg²⁺ binding to the DNAzyme is a cooperative process. Although the DNAzyme has five Hg²⁺ binding sites, the DNAzyme is stable enough to cleave its substrate after binding ˜2 Hg²⁺ ions. The detection limit was determined to be 2.4 nM based on 3σ/slope (inset of FIG. 3 b), which was a ˜16-fold improvement compared to the previous oligonucleotide folding based sensor.^([20]) Among all the reported Hg²⁺ sensors made from small and macro-molecules, this catalytic beacon has the best detection limit. The US EPA defined the toxic level of Hg²⁺ in drinking water to be 2 parts-per-billion or 10 nM, which can be covered by the beacon.

To test selectivity, the catalytic beacon responses in the presence of 13 competing metal ions were assayed (FIG. 4). Each metal was tested at three concentrations (1, 20, and 1000 μM). None of the metal ions gave responses higher than half of that produced by 20 nM Hg²⁺, and the selectivity was determined to be at least 100,000-fold higher for Hg²⁺ over any other metal ions (10 nM Hg²⁺ versus 1 mM competing metal ions).

Materials: All DNA samples were purchased from Integrated DNA Technologies Inc. (Coralville, IA, USA) and were purified by HPLC by the company. Uranium acetate dihydrate was purchased from Fisher Scientific (Hampton, N.H., USA) and other metal salts used in this work include: MgCl₂, CaCl₂, Mn(OAc)₂, Fe(NH₄)₂(SO₄)₂, FeCl₃, CoCl₂, NiCl₂, Cu(NO₃)₂, ZnCl₂, Cd(OAc)₂, Hg(ClO₄)₂, Pb(NO₃)₂, and TbCl₃.

Gel based assay: 1 μM 5′-FAM labeled 39S and 1 μM enzyme were annealed in 10 mM MES buffer pH 5.5 with 300 mM NaNO₃ by heating at 65° C. for 1 min and subsequently cooling slowly to room temperature in 30 min. For the experiment shown in FIG. 1 c, 8 μL of the annealed DNAzymes were placed in microcentrifuge tubes and 1 μL of 100 μM Hg(ClO₄)₂ was added. The system was allowed to incubate at room temperature for 10 min and 1 μL of 10 μM UO₂ ²⁺ was added to initiate the cleavage reaction. The reaction was stopped after 1 min by adding 10 μL of stop buffer containing 8 M urea and 50 mM EDTA. For E_(Hg)3T, E_(Hg4)T, E_(Hg)5T, and E_(Hg)6T DNAzymes, a 20 min time point was also taken for the samples without Hg²⁺, because the fractions of cleavage for these enzymes were too low at 1 min to allow accurate calculation. The reaction mixture was then loaded to a denaturing 20% polyacrylamide gel and the gel was imaged with a Molecular Dynamics STORM 840 fluorescence imager with excitation wavelength set at 450 nm.

Hg²⁺ detection: The catalytic beacon sensor was prepared by annealing 2 μM of the substrate and enzyme strand shown in FIG. 2 a in 10 mM MES, pH 5.5 and 300 mM NaNO₃. A large volume of the same buffer was also prepared. For each detection, 25 μL of the annealed sensor was mixed with 475 μL of buffer and the final sensor concentration was 100 nM. The fluorometer (Fluoromax-P, Horiba Jobin Yvon, Edison, N.J.) was set at the kinetics mode with 15 sec intervals. The excitation was set at 490 nm and emission at 520 nm. The temperature was set at 24° C. UO₂ ²⁺ (0.5 μL of 1 mM) was added to the DNAzyme to make a final concentration of 0.5 μM, and this mixture was used as the sensor for Hg²⁺. The sensor was vortexed and placed into the fluorometer to start the kinetics reading. After the first reading, the cuvette was taken out and a small volume of Hg(ClO₄)₂ or other metals was added. After vortexing, the cuvette was placed back to the fluorometer to continue the reading.

REFERENCES

-   (1) H. H. Harris, I. J. Pickering, G. N. George, Science 2003, 301,     1203. -   (2) United Nations Environment Programme Chemicals, Geneva,     Switzerland, 2002, p. 270. -   (3) L. Prodi, C. Bargossi, M. Montalti, N. Zaccheroni, N. Su, J. S.     Bradshaw, R. M. Izatt, P. B. Savage, J. Am. Chem. Soc. 2000, 122,     6769. -   (4) F. Szurdoki, D. Ren, D. R. Walt, Anal. Chem. 2000, 72, 5250. -   (5) X. Guo, X. Qian, L. Jia, J. Am. Chem. Soc. 2004, 126, 2272. -   (6) B. Liu, H. Tian, Chem. Comm. 2005, 3156. -   (7) A. Caballero, R. Martinez, V. Lloveras, I. Ratera, J.     Vidal-Gancedo, K. Wurst, A. Tarraga, P. Molina, J. Veciana, J. Am.     Chem. Soc. 2005, 127, 15666. -   (8) K. C. Song, J. S. Kim, S. M. Park, K.-C. Chung, S. Ahn, S.-K.     Chang, Org. Lett. 2006, 8, 3413. -   (9) Y. Zhao, Z. Lin, C. He, H. Wu, C. Duan, Inorg. Chem. 2006, 45,     10013. -   (10) W. Yang, Q. Hu, J. Ma, L. Wang, G. Yang, G. Xie, Journal of the     Brazilian Chemical Society 2006, 17, 1039. -   (11) M.-H. Ha-Thi, M. Penhoat, V. Michelet, I. Leray, Org. Lett.     2007, 9, 1133. -   (12) E. M. Nolan, S. J. Lippard, J. Am. Chem. Soc. 2003, 125, 14270. -   (13) Y.-K. Yang, K.-J. Yook, J. Tae, J. Am. Chem. Soc. 2005, 127,     16760. -   (14) S.-K. Ko, Y.-K. Yang, J. Tae, I. Shin, J. Am. Chem. Soc. 2006,     128, 14150. -   (15) E. M. Nolan, S. J. Lippard, J. Am. Chem. Soc. 2007, 129, 5910. -   (16) J. Wang, X. Qian, Org. Lett. 2006, 8, 3721. -   (17) J. Wang, X. Qian, J. Cui, J. Org. Chem. 2006, 71, 4308. -   (18) Y. Zhao, Z. Zhong, Org. Lett. 2006, 8, 4715. -   (19) Y. Zhao, Z. Zhong, J. Am. Chem. Soc. 2006, 128, 9988. -   (20) A. Ono, H. Togashi, Angew. Chem., Int. Ed. 2004, 43, 4300. -   (21) M. Virta, J. Lampinen, M. Karp, Anal. Chem. 1995, 67, 667. -   (22) M. F. Frasco, J.-P. Colletier, M. Weik, F. Carvalho, L.     Guilhermino, J. Stojan, D. Fournier, FEBS Journal 2007, 274, 1849. -   (23) M. Matsushita, M. M. Meijler, P. Wirsching, R. A. Lerner, K. D.     Janda, Org. Lett. 2005, 7, 4943. -   (24) P. Chen, C. He, J. Am. Chem. Soc. 2004, 126, 728. -   (25) S. V. Wegner, A. Okesli, P. Chen, C. He, J. Am. Chem. Soc.     2007, 129, 3474. -   (26) R. Vannela, P. Adriaens, Environmental Engineering Science     2007, 24, 73. -   (27) A. A. Vaughan, R. Narayanaswamy, Sens. Actuators B 1998, B51,     368. -   (28) X.-B. Zhang, C.-C. Guo, Z.-Z. Li, G.-L. Shen, R.-Q. Yu, Anal.     Chem. 2002, 74, 821. -   (29) B. Kuswandi, R. Narayanaswamy, Analytical Letters 1999, 32,     649. -   (30) B. Kuswandi, R. Narayanaswamy, Sens. Actuators B 2001, 874,     131. -   (31) W. H. Chan, R. H. Yang, K. M. Wang, Anal. Chim. Acta 2001, 444,     261. -   (32) A. Widmann, C. M. G. van den Berg, Electroanalysis 2005, 17,     825. -   (33) V. Ostatna, E. Palecek, Langmuir 2006, 22, 6481. -   (34) T. Balaji, M. Sasidharan, H. Matsunaga, Analyst 2005, 130,     1162. -   (35) J.-S. Lee, M. S. Han, C. A. Mirkin, Angew. Chem., Int. Ed.     2007, 46, 4093. -   (36) J. Li, Y. Lu, J. Am. Chem. Soc. 2000, 122, 10466. -   (37) J. Liu, A. K. Brown, X. Meng, D. M. Cropek, J. D. Istok, D. B.     Watson, Y. Lu, Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2056. -   (38) R. R. Breaker, Nat. Biotechnol. 1997, 15, 427. -   (39) Y. Lu, Chem. Eur. J. 2002, 8, 4588. -   (40) J. C. Achenbach, W. Chiuman, R. P. G. Cruz, Y. Li, Curr. Pharm.     Biotechnol. 2004, 5, 312. -   (41) G. F. Joyce, Ann. Rev. Biochem. 2004, 73, 791. -   (42) S. K. Silverman, Nucleic Acids Res. 2005, 33, 6151. -   (43) J. Liu, Y. Lu, J. Am. Chem. Soc. 2003, 125, 6642. -   (44) Y. Xiao, A. A. Rowe, K. W. Plaxco, J. Am. Chem. Soc. 2007, 129,     262. -   (45) Y. Miyake, H. Togashi, M. Tashiro, H. Yamaguchi, S. Oda, M.     Kudo, Y. Tanaka, Y. Kondo, R. Sawa, T. Fujimoto, T. Machinami, A.     Ono, J. Am. Chem. Soc. 2006, 128, 2172. -   (46) Y. Tanaka, S. Oda, H. Yamaguchi, Y. Kondo, C. Kojima, A.     Ono, J. Am. Chem. Soc. 2007, 129, 244. -   (47) J. Liu, Y. Lu, Meth. Mol. Biol. 2006, 335, 275. -   (48) M. Zuker, Nucleic Acids Res. 2003, 31, 3406. -   (49) R. R. Breaker, Curr. Opin. Biotechnol. 2002, 13, 31. -   (50) J. Liu, Y. Lu, Anal. Chem. 2003, 75, 6666. -   (51) W. Chiuman, Y. Li, Nucleic Acids Res. 2007, 35, 401. -   (52) G. A. Soukup, R. R. Breaker, Proc. Natl. Acad. Sci. U.S.A.     1999, 96, 3584. -   (53) R. P. G. Cruz, J. B. Withers, Y. Li, Chem. Biol. 2004, 11, 57. -   (54) Y. Lu, J. Liu, U.S. Pat. No. 6,890,719 (May 10, 2005). -   (55) Earnshaw & Gait, “Modified Oligoribonucleotides as     site-specific probes of RNA structure and function,” Biopolymers     (John Wiley & Sons, Inc.) 48:39-55, 1998. -   (56) Fluorescent and Luminescent Probes for Biological Activity. A     Practical Guide to Technology for Quantitative Real-Time Analysis,     Second Ed. W. T. Mason, ed. Academic Press (1999). -   (57) WO 99/27351. -   (58) Lakowicz, J. R. In Principles of Fluorescence Spectroscopy; 2nd     ed.; Kluwer Academic/Plenum: New York, 1999. 

1-32. (canceled)
 33. A nucleic acid enzyme, comprising: an oligonucleotide containing thymine bases, wherein the nucleic acid enzyme is dependent on both Hg²⁺ and a second ion as cofactors, to produce a product from a substrate comprising a ribonucleotide, a deoxyribonucleotide, or both.
 34. The nucleic acid enzyme of claim 33, wherein the oligonucleotide contains at least 4 thymine bases.
 35. The nucleic acid enzyme of claim 33, wherein the oligonucleotide contains at least 6 thymine bases.
 36. The nucleic acid enzyme of claim 33, further comprising a quencher.
 37. The nucleic acid enzyme of claim 33, wherein the second ion is selected from the group consisting of UO₂ ²⁺, Pb²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Co²⁺, Ni²⁺ and Mn²⁺.
 38. The nucleic acid enzyme of claim 33, wherein the second ion is UO₂ ²⁺.
 39. The nucleic acid enzyme of claim 33, wherein the oligonucleotide contains at least 38 bases.
 40. A method of detecting Hg²⁺ in a sample, comprising: forming a mixture comprising: (1) a nucleic acid enzyme, (2) the sample, (3) a substrate, and (4) a second ion, to produce a product from the mixture; and determining the presence of the product; wherein the enzyme comprises at least one quencher and is dependent on both the Hg²⁺ and the second ion as cofactors to produce the product from the substrate, the substrate comprises a ribonucleotide, a deoxyribonucleotide, or both, and the substrate comprises at least one fluorophore and at least one quencher.
 41. The method of claim 40, wherein the second ion is selected from the group consisting of UO₂ ²⁺, Pb²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Co²⁺, Ni²⁺ and Mn²⁺.
 42. The method of claim 40, wherein the second ion is UO₂ ²⁺.
 43. A method of determining the concentration of Hg²⁺ in the presence of other ions, in a sample, comprising: forming a mixture comprising: (1) a nucleic acid enzyme, (2) the sample, (3) a substrate, and (4) a second ion, to produce a product from the mixture; determining the presence of the product; and determining the concentration of the Hg²⁺ by measuring an amount of the product produced; wherein the enzyme comprises at least one quencher and is dependent on the Hg²⁺ and the second ion as cofactors to produce the product from the substrate, the substrate comprises a ribonucleotide, a deoxyribonucleotide, or both, and the substrate comprises at least one fluorophore and at least one quencher.
 44. The method of claim 43, wherein the second ion is selected from the group consisting of UO₂ ²⁺, Pb²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Co²⁺, Ni²⁺ and Mn²⁺.
 45. The method of claim 43, wherein the second ion is UO₂ ²⁺.
 46. A sensor for Hg²⁺, comprising: (1) a nucleic acid enzyme, (2) a substrate, and (3) a second ion, wherein the enzyme comprises at least one quencher and is dependent on both the Hg²⁺ and the second ion as cofactors to produce a product from the substrate, the substrate comprises a ribonucleotide, a deoxyribonucleotide, or both, and the substrate comprises at least one fluorophore and at least one quencher.
 47. The sensor of claim 46, wherein the nucleic acid enzyme comprises an oligonucleotide containing at least 2 thymine bases.
 48. The sensor of claim 47, wherein the oligonucleotide contains at least 4 thymine bases.
 49. The sensor of claim 47, wherein the oligonucleotide contains at least 6 thymine bases.
 50. The sensor of claim 46, wherein the second ion is selected from the group consisting of UO₂ ²⁺, Pb²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Co²⁺, Ni²⁺ and Mn²⁺.
 51. The sensor of claim 46, wherein the second ion is UO₂ ²⁺.
 52. The sensor of claim 47, wherein the oligonucleotide contains at least 38 bases. 